Comparison of Different Sample Preparation Protocols Reveals Lysis Buffer-Specific Extraction Biases in Gram-Negative Bacteria and Human Cells

We
evaluated different in-solution and FASP-based sample preparation
strategies for absolute protein quantification. Label-free quantification
(LFQ) was employed to compare different sample preparation strategies
in the bacterium Pseudomonas aeruginosa and human embryonic kidney cells (HEK), and organismal-specific
differences in general performance and enrichment of specific protein
classes were noted. The original FASP protocol globally enriched for
most proteins in the bacterial sample, whereas the sodium deoxycholate
in-solution strategy was more efficient with HEK cells. Although detergents
were found to be highly suited for global proteome analysis, higher
intensities were obtained for high-abundant nucleic acid-associated
protein complexes, like the ribosome and histone proteins, using guanidine
hydrochloride. Importantly, we show for the first time that the observable
total proteome mass of a sample strongly depends on the sample preparation
protocol, with some protocols resulting in a significant underestimation
of protein mass due to incomplete protein extraction of biased protein
groups. Furthermore, we demonstrate that some of the observed abundance
biases can be overcome by incorporating a nuclease treatment step
or, alternatively, a correction factor for complementary sample preparation
approaches.